The overall aim of my laboratory is to better understand the cellular and molecular underpinnings of the ontogeny of the mammalian hematopoietic system. We have recently found that primitive red cells in the embryo share significant similarities with their fetal and adult definitive counterparts, including an origin from unipotential progenitors, progressive maturational stages of nucleated erythroblasts, and terminal enucleation to become erythrocytes. However, primitive and definitive red cells also have significant differences in cell size, in transcriptional regulation, and in globin gene expression. We hypothesize that there are both shared and distinct stage-specific gene expression programs in differentiating primitive vs. definitive erythroid cells. In the first aim of this proposal, we will delineate a gene anatomy profile of primary primitive and definitive red blood cells at progressive stages of maturation. This data will be housed in a public database with a dedicated website for universal access. In the second aim, we will identify erythroid lineage-specific and stage-specific sets of genes and define erythroid-specific regulatory features using computationally assisted methods in collaboration with C. Stoeckert (U. Penn). The ability to compare similar stages of primitive red cells from the yolk sac and definitive red cells from the fetal liver and adult bone marrow gives us the unique opportunity to identify shared and differential programs of erythroid genes. In the third aim, we will test predictions generated in Aim 2 regarding the transcriptional regulation of lineage-specific and stage-specific gene cohorts. The function of specific candidate regulatory factors in erythroid differentiation will be screened by lentiviral siRNA-mediated gene knock-down in ES cell-derived primitive red cells. These studies will provide the research community with a comprehensive assembly of genes expressed at progressive stages of embryonic, fetal, and adult erythroid differentiation that will facilitate future characterization of structure-function relationships between expressed genes and red cell morphology, physiology, and disease phenotypes. Relevance of this research to public health. This research proposal is in response to a NIH program announcement (PA-03-150) to characterize all the genes in red blood cells. These studies will improve our understanding of the causes of anemia and will ultimately lead to curative treatments for children and adults with sickle cell disease and thalassemia syndromes.